The overall goals of this grant are to: a) understand the role of epigenetic gene silencing in cancer through studying the function of novel genes which have aberrant promoter hypermethylation; and b) dissect molecular mechanisms by which this promoter change participates in transcriptional silencing and through which it arises during cancer evolution. In the first specific aim, we have created a mouse knockout model for HIC-1 (Hypermethylated-in-Cancer-1), a gene cloned by screening for hypermethylated loci in a frequent LOH area for human cancers, 17p13.3. Hic-1 mice manifest an age dependent, gender specific, tumor spectrum and the wild type allele is lost in the tumors solely through promoter methylation. The loss of Hic-1 also profoundly alters tumor spectrum and aggressiveness in p53 mice including appearance of breast cancers. We will create new models for breast and other cancers based on this loss of Hic-1 function as a requisite early step. In the second specific aim, we have identified the developmental gene, ephrin A1, as a direct transcriptional repression target of HIC-1. We will study the significance of this for HIC-1 anti-tumor effects by: a) exploring whether ephrin AlsiRNA knockdown in human breast cancer cells reproduces effects of exogenous over-expression of HIC-1; and b) targeting overexpression of ephrin A1 to breast epithelium in a transgenic mouse. In the third specific aim, we identify that different histone modifications (the "histone code") mark promoter regions of transcriptionally silent and hypermethylated genes versus these same genes when they are unmethylated and expressed. Drug induced (5-deoxy-azacytidine-DAC) reversal of the hypermethylation leads to gene re-expression and loss of the transcriptional silencing histone marks and gain of transcriptional activation marks. We are exploring the hypothesis that the demethylation triggers these events through directly activating transcription which then prompts a nucleosome switch involving variant histones to account for loss of the silencing histone marks and gain of the transcriptional activation histone modifications. In specific aim # 4, we explore whether methylation of lysine 9 of histone H3 serves as a signal for triggering DNA methylation associated with cancer gene silencing. Our data in studies of a hypermethylated p16 gene in colon cancer ceils indicates that this may be the case. We will determine, through ChIP localization assays, and gene knockdown and over-expression studies, which specific methyl K9:H3 methyltransferases may trigger this histone modification and thus may establish aberrant promoter DNA hypermethylation in cancer cells. A final specific aim links all of those above by exploring the hypothesis that specific chromatin changes may explain the gender differences in the spectrum of tumors seen in mice with disruption of the gene being studied in Specific Aims #'s 1 and 2.